Estrogen receptor-1 (ER) and tumor suppressor p53 play important, but opposite, roles in the onset and progression of breast cancer. Compared to other cancers, overall frequency of p53 mutation in breast cancer is about 20%; however, wild type p53 is functionally debilitated. Both ER and p53 are localized in the nuclei as well as mitochondria and are functionally important in both the compartments. ER has been reported to bind and inhibit wild type p53 function in the nucleus. There is a fundamental gap in understanding how mitochondrial p53 is antagonized by ER. This gap represents an important problem because deciphering the role of ER in suppressing mitochondrial p53 is essential in understanding the mechanisms by which wild type p53 is inactivated in breast cancer. The long-term goal is to understand the mechanisms by which nuclear and mitochondrial functions of p53 are compromised in breast cancer. The objective in this particular application is to analyze mitochondrial ER-p53 interaction and its functional consequences. The central hypothesis is that ER and p53 interact within mitochondria leading to important functional consequences. The rationale that underlies the proposed research is that given the importance of mitochondrial p53 in responding to oncogenic signaling and regulation of cellular metabolism, understanding the interaction between mitochondrial ER and p53 and its functional consequences would provide new therapeutic targets in addition to mechanistic insights that could be exploited for better intervention strategies. This hypothesis wil be tested by pursuing three specific aims: 1) Analyze ER-p53 interaction in mitochondria in breast cancer cells; 2) Investigate effect of ER- p53 interaction on oxidative phosphorylation (OXPHOS) in mitochondria; and 3) Determine the effect of ER- p53 interaction on mitochondrial gene transcription and apoptosis. Under the first aim, already proven immunoprecipitation (IP) assay, RNA interference (RNAi) approach, and gene transfection approaches, which have been established in the applicants' laboratories, will be used to characterize ER-p53 interaction within mitochondria. Under the second specific aim, effect of ER-p53 interaction on OXPHOS will be analyzed using approaches such as respiratory complex assays and quantitative real-time PCR (qPCR) assay that are established in the applicants' laboratories along with a OXPHOS-Chip expression array already developed and validated by the applicants. The third aim will address, using multiple technical approaches already optimized by the applicants, how ER-p53 interaction affect mitochondrial gene transcription and apoptosis. The research is innovative because it represents a new and substantive departure from the status quo, namely analyzing ER and p53 function in breast cancer cell mitochondria in an integrated manner instead of pursuing them as independent regulators of separate pathways. The proposal is significant because it is the first step in a continuum of research that is expected to lead to a better understanding of the roles of mitochondrial ER and p53 in breast cancer that could be exploited for developing new therapeutic strategies.